In this continuing Bioengineering Research Partnership project, we seek to design improved antibody drugs for blocking aberrant signaling by epidermal growth factor receptor (EGFR) in cancer. A team with expertise in protein engineering (Wittrup, MIT), structural biology (Kuriyan, UC Berkeley), cell engineering (Lauffenburger, MIT), proteomics (White, MIT), and medical imaging (Larson, MSKCC) will further develop and characterize a panel of anti-EGFR molecules isolated in the previous project period. Our central hypothesis is that antibodies binding to domain IV of EGFR (anti-IV) block signaling in a ligand-independent manner, enabling antagonism of a common truncation mutant (EGFRvlll) that is not antagonized by the current generation of anti-EGFR antibodies. Anti-IV antibodies and fluorescence-based biophysical techniques will be used to further study the transduction of a symmetric ligand-bound extracellular dimerization event to an asymmetric active kinase dimer in the cytoplasm, recently characterized as part of this project. The molecular allosteric mechanisms that result in kinase domain activation upon ligand binding will be delineated so as to understand better how to block this process. We will apply mass spectrometry to simultaneously quantify the phosphorylation kinetics of hundreds of specific tyrosines in the signaling pathways downstream of EGFR, in particular looking for proximal evidence for eradication of EGFRvlll's effects by anti-IV antibodies. Enhanced receptor downregulation with multivalent antibodies will be systematically studied and exploited to reduce receptor number. Effects of anti-IV antibodies on migration and proliferation will be examined in autocrine cell lines. The kinetics of antibody penetration into tumor spheroids will be studied by 2-photon microscopy. In mouse xenograft models, the pharmacokinetics and pharmacodynamics of these antibodies will be studied by PET. This project is a systematic effort by a team of engineers, scientists, and physicians to develop a new generation of anticancer drugs through improved mechanistic understanding and blocking of a common class of uncontrolled growth signals sent by receptors on the surface of cancer cells.